Method of recloning production cells

ABSTRACT

A new method for selecting clones and recloning mammalian cells which are of importance for the production of biopharmaceuticals, preferably hamster or mouse myeloma cells, with a high degree of automation and throughput. The invention relates to methods of depositing and replicating single cell clones of the cells in question. The invention also relates to methods of preparing proteins using cells which have been obtained and replicated by single cell deposition as well as compositions which allow the replication of single cells.

RELATED APPLICATIONS

This application claims priority benefit of U.S. Ser. No. 60/498,463,filed Aug. 28, 2003, and German Application No. 103 38 531, filed Aug.19, 2003, each of which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the field of cell culture technologyand concerns methods for replicating/cloning cells, preferably celllines, which are of importance for the production of biopharmaceuticals.The invention also relates to methods for preparing proteins using cellswhich have been obtained and replicated by individual cell depositing,and also compositions which allow replication of individual cells.

BACKGROUND TO THE INVENTION

For the biotechnological production of biologically active ortherapeutic proteins in mammalian cells, so-called biopharmaceuticals,the corresponding mammalian cells are stably transfected with DNA whichcodes for the biologically active protein (or its subunits). After thetransfection process a pool of millions of differently transfected cellsis normally obtained. Therefore the crucial step for the preparation ofefficient production cell lines is in the selection and replication ofcell clones which on the one hand grow very stably and on the other handshow a high specific productivity of therapeutic protein (productformation etc.). As there are millions of different product-expressingcells, it is critical to be able to analyse a plurality of cellsindividually with a high throughput and to use automation in order to beable to sort out suitable candidates (single cell clones) which bothgrow very robustly and also yield high product titres. This process ofsingle cell isolation and subcultivation is known as cloning orrecloning.

Transfected cells may be selected by fluorescence-activated cell sorting(FACS) for example, by linking the expression of the therapeutic proteinto the expression of a marker protein. For this purpose for examplefluorescent proteins and the variants thereof of Aequorea victoria,Renilla reniformis or other species, including but not limited to thered, yellow, violet, green fluorescent proteins or the variants thereofof non-bioluminescent organisms such as e.g. Discosoma sp., Anemoniasp., Clavularia sp., Zoanthus sp. may be co-expressed in a cell togetherwith the therapeutic protein. Conclusions may be drawn from thefluorescence intensity as to the specific productivity and the growthcharacteristics of the cells.

However, there is the problem of depositing typical recombinantproduction cells such as mouse myeloma (NS0), hamster ovary (CHO), orhamster kidney cells (BHK), particularly if they are adapted to growthin serum-free suspension cultures, i.e. under modern production-relevantcell culture conditions, individually in culture vessels, e.g. in thewells of microtitre plates, under serum-free culture conditions, andeffectively replicating (recloning) them. If only a few cells, forexample less than 5 cells, are deposited in a culture vessel underserum-free conditions, these cells cannot replicate at all, or at leastcannot replicate efficiently. The reason for this is suspected to be theabsence of cell-to-cell contacts, a greater nutrient/growth factorrequirement at a lower cell density and/or the absence or very lowconcentration of diffusible signal and conditioning factors.

In the prior art the problem of serum-free single cell cloning in theabove-mentioned recombinant production cells is avoided by generatingcell clones by the limited dilution method. In this, a minimum of 5 to10 cells are seeded in serum-free medium in a culture dish and thensubpassaged by repeated dilution cloning in order to obtain, instatistical terms, a culture consisting of genetically identical cells(method=limited dilution). On the one hand this method of recloning istime-consuming and on the other hand it usually leads to geneticallyheterogeneous mixed cultures, as the process is based on a statisticalcalculation and not on actual cultivation of individually depositedgenetically identical cells. These heterogeneous mixed cultures aregenerally characterised by limited robustness with respect tofermentation and a heterologous expression profile.

Alternatively, at present, single cell clones of production-relevantcell lines can only be generated by the individual depositing ofserum-adapted adherent cells. Thus, Meng et al., (Meng Y G., et al.,Gene 2000, 242, 201-207) mention, for example, a method of depositingindividual, adherently growing CHO cells in serum-containing medium. Themethod described by Meng et al., however, has major disadvantages; i.e.,because of the adherence of the cells, the laborious enzymaticdetachment of the cells from the substrate (trypsin treatment) may leadto considerable cell damage and changes in the growth characteristicsand in the productivity of the recloned cells. Moreover, thecorrespondingly obtained individual clones then have to be adapted toserum-free growth in suspension culture, which is normally atime-consuming operation and affects the productivity of the cells andthe product quality (cf. on this subject, inter alia, Kaufmann H. etal., Biotechnology and Bioengineering 2001, 72, 592-602; Mueller et al.,Biotechnology and Bioengineering 1999, 65, 529-532).

By using nutrient cells, also known as feeder cells, in the cultivationof adherently growing cells, it is possible to influence the growthcharacteristics of cells for the better or, for some types of cell, toreplicate them under cell culture conditions for the first time.Examples include human-mouse or mouse-mouse hybridoma cells (Hlinak A.et al., Folia Biologica (Praha) 1988, 34, 105.117, U.S. Pat. No.5,008,198), primary keratinocytes (Rheinwald, J G and Green, Cell 1975,6, 331-344; WO 9954435), stem cells (Williams R L., et al., Nature 1988,336, 684-687) and various tumour cells (Wee Eng Lim et al., Proteomics2002, 2, 1187-1203; Rexroad et al., Molecular Reproduction andDevelopment, 1997, 48, 238-245; Peng et al., Biotechnology andBioengineering, 1996, 50, 479-492; Grigoriev et al., AnalyticalBiochemistry, 1996, 236, 250-254; Sanchez et al., Journal ofImmunological Methods, 1991, 145, 193-197; Butcher et al., Journal ofImmunological Methods, 1988, 107, 245-251; Long et al., Journal ofImmunological Methods, 1986, 86, 89-93; Shneyour et al., Plant ScienceLetters, 1984, 33, 293-302; Pintus et al., Journal of ImmunologicalMethods, 1983, 61, 195-200; Brodin et al., J Immunological Methods,1983, 60(1-2), 1-7). Feeder cells are usually cells the growth of whichhas been chemically or physically arrested, which have lost theircapacity for cell division as the result of a special pre-treatment butotherwise still remain vital for about 2 to 3 weeks on average. Feedercells are thus still capable of releasing growth-promoting factors intothe medium and can thus promote the initial growth of non-arrested cellsor even make this growth possible, in the case of various primary cells.For this purpose the feeder cells are plated out in a culture dish as aso-called monolayer. Then the adherently growing cells which are to becultivated are plated out on or between the feeder cells and cultivatedunder standard conditions. Feeder cells may be prepared for example byirradiation or treatment with mitomycin C(azirino[2′,3′:3,4]pyrrolo[1,2-a]indole-4,7-dione,6-amino-8-[[(aminocarbonyl)oxy]methyl]-1,1a,2,8,8a,8b-hexahydro-8a-methoxy-5-methyl-,[1aR-(1a.alpha., 8.beta., 8a.alpha., 8b.alpha.)]-(9Cl) (Butcher et al.,Journal of Immunological Methods, 1988, 107, 245-251)). Primary cellssuch as spleen cells, fibroblasts, blood cells (Morgan, Darling; Kulturtierischer Zellen [culture of animal cells]. Spektrum AkademischerVerlag 1994, p. 115f) and macrophages (Hlinak A. et al., Folia Biologica(Praha) 1988, 34, 105.117) have been described in feeder cell systems.In connection with the production of antibodies in hybridoma cells theuse of feeder cells and FACS-based cell selection was described. Hlinaket al. (1988) for example describe a recloning efficiency of 33 to 57%in the recloning of adherently growing hybridoma cells starting from two(2) cells per culture dish. The hybridoma cells used were adherentlygrowing cells adapted to serum-containing medium.

When using heterologous, particularly human feeder cells for generatingproduction cells there is a considerable risk of contamination bypathogens such as, for example, viruses, bacteria or mycoplasms.Moreover, many of the (primary) feeder cells described requireserum-containing medium, which firstly increases the risk ofcontamination and secondly has the drawback that production cells whichhave laboriously been adapted to serum-free growth have to bere-adapted.

When using heterologous feeder cells, it is generally necessary tocounter-select the feeder cells. Production cells are generally subjectto a selection pressure, e.g. as a result of the use of additives to themedium (antibiotics such as G418) and/or incomplete media (absence ofhypoxanthine, thymidine). This selection pressure makes it possible toselect cells, for example, which have absorbed and integrated thecorresponding genetic information for a recombinant protein. The mediumthus produced which is adapted to the production cell criticallyinfluences the growth of the feeder cells and together with theotherwise incompatible production medium leads to a very rapid dying offof the feeder cells. As a result, the function of the feeder cells is nolonger guaranteed.

SUMMARY OF THE INVENTION

One aim of the present invention was to find an efficient recloningmethod which allows production-relevant mammalian cells to be replicatedunder serum-free conditions and in suspension culture, starting fromless than five cells, preferably from one (1) single cell. Inparticular, the aim was to provide corresponding processes for recloningCHO or BHK cells originally isolated from hamsters and myeloma cellsoriginally isolated from mice, e.g. NS0 cells.

A further aim of the invention was to provide compositions which make itpossible to carry out the corresponding recloning methods, particularlythose for recloning hamster or mouse myeloma cells.

These objectives are achieved by means of the objects according to theinvention as defined in the patent claims, which according to one aspectof the invention relates to a method for cloning cells, characterised inthat fewer than five, preferably one (1) or two (2) mammalian cell(s),preferably hamster or mouse myeloma cells, are deposited in the presenceof feeder cells, preferably of autologous origin, in a culture vesselunder serum-free conditions and cultivated and replicated underserum-free conditions. One particular aspect of the invention relates toa corresponding process for recloning CHO or BHK cells (hamster cells)or NS0 cells (mouse myeloma cells), preferably when the cells are thosewhich are adapted to serum-free growth in suspension cultures.

Another embodiment of the invention relates to the use of hamster cellsas feeder cells in the event that the mammalian cells which have beendeposited and are to be cloned are also hamster cells, particularly CHOor BHK cells. Mouse myeloma cells are preferably used as feeder cells ifthe cells which have been deposited and are to be cloned are NS0 cells.

In another aspect, the invention relates to a corresponding process forrecloning CHO, BHK or NS0 cells, which is characterised in that CHOcells are used as feeder cells if the mammalian cells to be cloned areCHO cells, in that BHK cells are used as feeder cells when the mammaliancells to be cloned are BHK cells, and that NS0 cells are used as feedercells when the mammalian cells to be cloned are NS0 cells.

The processes according to the invention are characterised by a goodrecloning efficiency of more than 10%, preferably more than 20%,particularly for individually deposited cells. According to anotherembodiment the recloning methods according to the invention have arecloning efficiency of more than 30%, preferably more than 40%,particularly preferably more than 50%, more preferably more than 60%,even more preferably more than 70%, yet more preferably more than 80%.

In this case recloning efficiencies from 10 to more than 65% (e.g., atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%) in the recloning of individually deposited CHO cells, from 10to more than 50% (e.g., at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%) in the recloning of individually deposited BHKcells and from 10 to more than 45% (e.g., at least 10%, at least 20%, atleast 30%, at least 40%) in the recloning of individually deposited NS0cells, are regarded as efficient. If more than one (1) cell is depositedper culture dish, for example two, three or four, the recloningefficiency for the cells in question is above the values specified forthe recloning of CHO, BHK and NS0 cells.

In another aspect the invention relates to a corresponding process forrecloning mammalian cells, particularly hamster or mouse myeloma cells,wherein the cells to be cloned are replicated in the presence of 100 to200,000 feeder cells per ml of medium.

The present invention also relates to processes for producing proteins,preferably recombinant proteins, in serum-free non-adherently growingmammalian cells which have been grown by one of the cloning methodsaccording to the invention, particularly the production of recombinantproteins in correspondingly cloned hamster or mouse myeloma cells suchas for example CHO, BHK or NS0 cells, under serum-free conditions,comprising the steps of:

-   -   a) culturing mammalian cells which express a gene product of        interest, under serum-free conditions, which allow the cells in        question to replicate;    -   b) depositing less than five (5), preferably one (1) or two (20        of the corresponding mammalian cells in one cell culture vessel        under serum-free conditions;    -   c) replicating the correspondingly deposited cells in the        presence of autologous feeder cells under serum-free conditions;    -   d) cultivating the replicated deposited cells under serum-free        conditions under which the gene of interest is expressed; and    -   e) recovering and purifying the gene product which is coded by        the gene of interest from the cells including the membrane or        from the culture supernatant.

The expression of a recombinant gene product also requires thetransfection of the mammalian cells with a nucleic acid which codes forthe gene product of interest.

The mammalian cells can, for example, be separated manually or byFACS-based sorting and deposited in a cell culture vessel with feedercells. Preferably non-adherently cultivated feeder cells are used.

The present invention also relates to the fundamental use of hamstercells, preferably CHO or BHK cells and mouse myeloma cells, preferablyof type NS0, as feeder cells. According to a preferred embodiment, thepresent invention relates to the use of corresponding cells adapted toserum-free culture conditions.

The present invention also relates to compositions consisting of aserum-free cell culture medium, fewer than five mammalian cells capableof dividing and feeder cells autologous with the divisible mammaliancells. According to a further aspect of the invention, the divisiblemammalian cells are cells which are adapted to serum-free growth as asuspension culture. In a particular embodiment the composition containsonly one (1) or two (2) mammalian cells capable of dividing in theculture medium. The divisible mammalian cells are preferably hamstercells such as for example CHO or BHK cells, or mouse myeloma cells, e.g.NS0 cells.

According to another aspect of the invention, the composition containshamster cells, preferably CHO cells as feeder cells if the divisiblemammalian cell(s) are CHO cells. If the divisible mammalian cell(s) areBHK cells, the composition also contains hamster cells, but preferablyBHK cells as feeder cells. If the divisible mammalian cell(s) are mousemyeloma cells, for example NS0 cells, the composition also containsmouse myeloma cells as feeder cells, and in the case of NS0 cellspreferably also contains NS0 cells as feeder cells.

Surprisingly, it has been found that by using suspended autologousfeeder cells, single mammalian cells, preferably hamster or mousemyeloma cells, can be deposited in serum- and/or protein-free medium andgrow into cultures and thus the above-mentioned disadvantages ofrecloning, for example by limited dilution, or the adaptation ofmonoclonal cell lines to serum-free growth in suspension culture can beovercome.

The process according to the invention makes it possible to depositcorresponding mammalian cells in cells in serum-free, protein-free orchemically defined production medium, so that there is no need to carryout adaptation to serum-free or protein-free production medium, as wouldnormally be required. This leads to a substantial shortening ofdevelopment times (about 50%) in establishing production-relevant celllines. Moreover, the deposition of single cells leads to stable,homogeneous cell clones, which is of crucial importance in theproduction of biopharmaceuticals, not least with regard to regulatoryrequirements in their licensing as drugs. Furthermore, the use ofautologous feeder cells for the recloning of production-relevant CHO,BHK or NS0 cells, e.g. the use of hamster cells, preferably CHO or BHKcells, or mouse myeloma cells, preferably NS0 cells, has a considerablylower risk of contamination with regard to human pathogens than the useof human or less well-characterised cells.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the correlation between the dose of energy used in theproduction of the feeder cells and the cloning efficiency in therecloning of automatically deposited single CHO-DG-44 cells. The cellsare deposed in each case onto about 2000 inactive autologous feedercells. The graphics show that feeder cells irradiated with an energydose of 20-500 Gy still release enough factors into the medium to allowmore than 65% of the single clones deposited at 50 Gy to grow intocolonies.

FIG. 2 shows the productivity of antibody-expressing CHO-DG-44 cultureswhich have been obtained by limited dilution (left-hand column) orsingle cell deposition (right-hand column). The left-hand column showsthe productivity of 6 cultures which have been obtained by limiteddilution. The right-hand side shows the productivity of 6 single clonesfrom the automated single cell deposition. To determine theproductivity, three parallel experiments were carried out per cellclone/culture. In contrast to the cloning by limited dilution, cloningby automated deposition of single clones led to a substantially lowervariation in the productivity. The subcultures cultivated in paralleland derived from a single clone exhibit substantially higher homogeneitywith regard to their productivity compared with the subcultures obtainedby limited dilution.

FIG. 3 a shows the product titre of CHO-DG44 cell clones which have beensorted according to various criteria and individually deposited. In thebottom graph, single cells which meet only the sorting criterion “livingcell” have been deposited. This criterion was defined using theForward-Side-Scatter recording in the Flow Cytometer. The cells have notbeen selected by fluorescence. On the other hand, in the central and topgraphs B and C the sorting criterion “fluorescence of the cells” hasadditionally been logically linked to the sorting criterion “livingcell”. The sorting criterion “fluorescence of the cells” was alsofurther defined by means of the fluorescence intensity. To do this, onthe one hand the top 20% fluorescent cell clones and on the other handthe top 5% fluorescent cell clones were individually deposited. Theshift to the right in the histogram C shows that the proportion ofhigh-expressing cell clones when using the criterion of the top 5%fluorescent cells increases significantly compared with the othersorting criteria used in A and B.

FIG. 3 b is based on the data shown in FIG. 3 a. The percentageprobability of obtaining a producer with double the average productivity(=high producer) is shown. A normal distribution was matched to the dataobtained for all the living cells and the average titre was determined.Then, the percentage at which the cell clones obtained by individualcell deposition have double the titre or more was calculated from thedistribution function for normal distribution. This percentage is shownin FIG. 3 b. The graph shows that the probability of a high producerwhen additionally using the top 5% criterion compared with the use ofthe criterion “living cell” on its own increases more than twenty-fold.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Before the more detailed description of the invention by means of thenon-restrictive exemplifying embodiments that follow, it should bepointed out that the use of the indefinite article, for example “a” or“an” and the definite article, namely “the”, includes both the singularand plural of the term in question, unless one of the two forms isexplicitly ruled out and reference is made to a particular form(singular or plural). Thus, the term “a cell” automatically includes “aplurality of cells” as well, unless it is explicitly stated that only asingle cell is meant. The singular is explicitly meant, for example,where “a” or “one” is supplemented by (1).

Definitions

The term “cloning/recloning”, “clone/recline” in connection with cellculture means a technique by means of which a cell population ofidentical cells can be obtained from an original cells. The term “cellcloning” or “single cell cloning” thus means a process wherein singlecells can be identified and isolated from a cell pool with cells ofdifferent genotypes and then replicated to form a cell populationconsisting of a plurality of genetically identical cells. If the cellsare deposited individually, i.e. only one (1) cell per culture vessel,and then expanded to form a cell population of identical cells, theprocess is “direct single cell cloning”. If a number of cells aresimultaneously deposited in a culture vessel, expanded to form a cellpopulation and this is divided up into cell populations of identicalcells by repeated dilution (=limited dilution), this is described as an“indirect cloning” method.

Single clones are genetically identical cells which originate from one(1) single cell. A cell population consisting of identical cells of thesame origin is consequently referred to hereinafter as a “monoclonalcell population”. If during the cultivation of cells of the same originthere are spontaneous changes in the genome, for example mutationsand/or translocations, the individual cells of this cell population arestill regarded as identical cells for the purposes of the presentinvention, and the culture is regarded as a monoclonal cell population.By contrast, a pool of stably transfected cells (transfectants) are notcell clones of the same lineage, i.e. they are not a monoclonal cellpopulation, even if genetically identical starting cells are transfectedwith an identical nucleic acid.

The term “subclones/subcultures” refers to different generations ofcells which are produced from an original cell or original culture bysingle or multiple passaging of the dividing cells. The words“subclones/subcultures” are used, for example, when identical cells orcell cultures are cultivated and replicated over a number ofgenerations.

By “effective or efficient recloning” is meant a cloning efficiency ofat least 10%, preferably at least 20%, more preferably at least 30% andeven more preferably at least 40%. According to a particularly preferredembodiment of the present invention the term effective recloning meanscloning with an efficiency of at least 50%, preferably at least 60%,most preferably at least 70% and even more preferably at least 80%.

The term “cloning efficiency” is defined as the percentage of cellswhich can form vital cell populations of preferably more than 50 cellsafter being deposited. If for example in a cell sorting operation 50cells are distributed over 50 culture vessels and if 25 of these 50individually deposited cells grow to form cultures, the cloningefficiency is 50% (25 out of 50).

The term “capable of division/expandable” for the purposes of thepresent invention describes the potential of a cell/cell population todivide endlessly but at the least over 2, preferably 4, passages. Thispotential may for example by reduced or destroyed altogether byirradiation with ^([137])Cs or by mitomycin C treatment.

The term “derivative/descendant” refers to cells which can be tracedback genetically to a particular starting cell and are formed forexample by subcultivation (with or without selection pressure) and/orgenerated by gene manipulation. Re-isolations of cells of the same celltype are also included in the term “derivative/descendant”. Thus, forexample, all CHO cell lines are derivatives/descendants of the hamsterovary cells isolated from Cricetulus griseus by Puck et al., 1958,regardless of whether they were obtained by subcultivation, re-isolationor gene manipulations.

The term “autologous feeder cell” means that both the feeder cell andthe cell which is to be cultivated in the presence of this feeder cellare derived taxonomically from the same origin. If for example the cellto be cultivated is a hamster cell (subfamily Cricetinae), preferably acell of the genus Cricetulus or Mesocricetus, for example a CHO or BHKcell, each feeder cell originally isolated from this subfamily is afeeder cell which is autologous to these hamster cells of the subfamilyCricetinae. According to a preferred embodiment the term “autologousfeeder cell” means that both the feeder cell and the cell which is to becultivated were derived from the same genus taxonomically or wereoriginally isolated from the same genus (cells from Cricetulus orMesocricetus). If for example the cell to be cultivated is a hamstercell of the genus Cricetulus or Mesocricetus, preferably a CHO or BHKcell, each feeder cell originally isolated from the genus in question isan autologous feeder cell in the sense of this invention. According toanother preferred embodiment an autologous feeder cell is present if thefeeder cell and the cell to be cultivated come from the same species,for example Cricetulus griseus or Mesocricetus auratus. According to aparticularly preferred embodiment an autologous feeder cell is presentif both the feeder cell and the cell to be cultivated come from the samespecies and have the same tissue tropism (e.g. ovarian cells fromCricetulus griseus—CHO cells). According to a particularly preferredembodiment, a feeder cell is an autologous feeder cell if both thefeeder cell and the cell to be cultivated originate from the same basiccell, for example if both cells were originally CHO-DG-44 cells ordescendants of these cells. According to another preferred embodimentthe feeder cell confers the same resistances, e.g. to antibiotics, asthe cell which is to be cultivated. This is particularly advantageouswhen the cell deposition is carried out in the presence of a selectingagent, e.g. an antibiotic.

The term “serum-free” means culture media and also cultivationconditions which are characterised in that cells are grown in theabsence of animal and/or human serum, preferably in the absence of anyproteins isolated from serum, preferably in the absence ofnon-recombinantly produced proteins. Consequently, the term “cellsadapted to serum-free conditions” means those cells which can bereplicated in the absence of animal or human serum or serum proteins.

The term “protein-free” means that the culture medium does not containany animal proteins; proteins isolated from bacteria, yeasts or fungiare not regarded as animal proteins.

The term “chemically defined” describes a cell culture medium which isserum-free, preferably also protein-free, and which consists ofchemically defined substances. Chemically defined media thus consist ofa mixture of predominantly pure individual substances. One example of achemically defined medium is the CD-CHO medium produced by MessrsInvitrogen (Carlsbad, Calif., US).

The expression “a cell which may be cultivated in suspension” refers tocells which are adapted to growth in liquid cultures (“suspensioncultures”) and whose ability to adhere to the surfaces of vessels, forexample cell culture dishes or flasks, has been restricted or lost.Cells which are adapted both to serum-free growth and to growth insuspension are referred to as “non-adherent cells adapted to serum-freemedium”. If feeder cells are prepared from such cultures, these cellsare by definition “non-adherent feeder cells adapted to serum-freemedium”.

DESCRIPTION OF THE INVENTION

The present invention relates to a method of cloning cells,characterised in that fewer than five, e.g. four, three, two or one (1)mammalian cell(s) is or are deposited in a culture vessel in thepresence of feeder cells, preferably autologous feeder cells, in aculture vessel under serum-free conditions and cultivated and replicatedunder serum-free conditions. According to a preferred embodiment thepresent invention relates to a corresponding method of cloning mammaliancells, characterised in that one (1) or two mammalian cell(s) perculture vessel is or are deposited under serum-free conditions andcultivated in the presence of autologous feeder cells under serum-freeconditions. A preferred embodiment relates to a method of cloning singlecells, characterised in that one (1) single mammalian cell is depositedin a culture vessel in the presence of autologous feeder cells underserum-free conditions, and cultivated and replicated under serum-freeconditions. In another preferred embodiment the deposited cell which isto be cultivated is a cell growing in suspension culture.

If only one (1) cell is deposited per culture vessel and replicated toform a cell population, each individual growing cell population is amonoclonal cell population and the process is a method of direct singlecell cloning. If more than one (1) single cell is deposited andreplicated in each culture vessel, for example two, three or four cells,the growing cell populations are so-called mixed clones. These may thenbe converted into so-called statistical monoclonal cell populations bydirect single cell cloning or by conventional methods, for example byrepeated dilution of the cell populations (=limited dilution) (see forexample Morgan, Kultur tierischer Zellen [culture of animal cells],pages 113 and 114, Spektrum Akademischer Verlag 1994).

The processes provided by the present invention can be used particularlyto replicate and clone mammalian cells of the subfamily Murinae, forexample of the genus Mus or subfamily Cricetinae, for example of thegenera Cricetulus or Mesocricetus, as well as cell lines isolated fromthem, including their descendants/derivatives. Particularly preferred isthe method of replicating/cloning hamster cells or mouse myelomaaccording to the invention and stable cell lines derived therefrom.Accordingly, the present invention relates to a method of cloning cellsin the presence of feeder cells, preferably autologous feeder cells,characterised in that the deposited mammalian cells which are to becloned are hamster or mouse myeloma cells.

According to a particularly preferred embodiment the processes accordingto the invention are methods of replicating/cloning hamster cells of thegenus Cricetulus (Chinese dwarf hamster) and stable cell lines isolatedfrom this genus or derived from the isolated cells, e.g. CHO, CHO-K1,CHO-DUKX, CHO-DUKX B1 or CHO-DG-44 cells and derivatives/descendants ofthese cell lines. Particularly preferred according to the invention is aprocess wherein CHO-DG-44, CHO-DUKX, and CHO-K1, particularly CHO-DG-44and CHO-DUKX cells are replicated and cloned in the presence ofautologous feeder cells. Using the processes according to the invention,cells from Mesocricetus auratus (Syrian hamster) and stable cell linesisolated therefrom or derived therefrom, for example BHK21 or BHK TK⁻cells and derivatives/descendants of these cell lines may also bereplicated and cloned by the processes described herein. Consequently,the present invention preferably relates to a method of replicating andcloning CHO or BHK cells, and the derivatives/descendants thereof,characterised in that fewer than five, for example four, three, two, orpreferably only one (1) cell(s) is or are deposited in a culture vesselin the presence of autologous feeder cells under serum-free conditionsand cultivated and replicated under serum-free conditions.

Moreover the present invention relates to a method of replicating andparticularly cloning mouse myeloma cells, preferably from Mus musculusand stable cell lines isolated or derived therefrom, for example NS0 andSp2/0 cells and derivatives/descendants of these cell lines. Thisprocess is also characterised in that fewer than five, for example four,three, two, or preferably only one (1) of these cells is or aredeposited in a culture vessel in the presence of autologous feeder cellsunder serum-free conditions and cultivated and replicated underserum-free conditions.

Additional Examples of hamster and mouse cells which may be replicatedand cloned according to the invention are specified in the followingTable 1. In addition to derivatives and descendants of these cells/celllines, other mammalian cells, including cell lines from humans, mice,rats, monkeys, or rodents other than mice and hamsters may be replicatedor cloned by a process according to the invention.

TABLE 1 Hamster and mouse cell lines cell line Accession number NS0ECACC No. 85110503 ATCC CRL1827 ATCC CRL2695, 2696 Sp2/0Ag14 ATCCCRL1581 BHK21 ATCC CCL10 BHK TK⁻ ECACC No. 85011423 HaK ATCC CCL15225462.2 (BHK21 Derivative) ATCC CRL8544 CHO ECACC No. 8505302 CHO-K1ATCC CCL61 CHODUKX ATCC CRL9096 (= CHO duk⁻, CHO/dhfr⁻) CHODUKX B1 ATCCCRL9010 CHODG44 Urlaub et al., Cell 33[2], 405412, 1983 CHO Pro5 ATCCCRL1781 V79 ATCC CCC93 B14AF28G3 ATCC CCL14 CHL ECACC No. 87111906

According to the invention the corresponding mammalian cells arepreferably established, cultivated and deposited under serum-freeconditions. These steps are optionally carried out in media which arefree from animal proteins/peptides and/or chemically defined. Examplesof commercially obtainable media include Ham's F12 (Sigma, Deisenhofen,DE), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM;Sigma), Minimal Essential Medium (MEM; Sigma), Iscove's ModifiedDulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, Calif.,USA), CHO-S-SFMII (Invitrogen), serum-free CHO-medium (Sigma) andprotein-free CHO-medium (Sigma). Each of these media may optionally besupplemented with various compounds, e.g. hormones and/or other growthfactors (e.g. insulin, transferrin, epidermal growth factor,insulin-like growth factor), salts (e.g. sodium chloride, calcium,magnesium, phosphate), buffers (e.g. HEPES), nucleosides (e.g.adenosine, thymidine), glutamine, glucose or other equivalent nutrients,antibiotics and/or trace elements. In order to select geneticallymodified cells which express one or more selectable marker genes, one ormore suitable selecting agents, e.g. antibiotics, may be added to themedium.

Up till now, the use of feeder cells for cultivating cells has beendescribed in connection with the cultivation of adherently growing cells(e.g. Wee Eng Lim et al., Proteomics 2002, 2, 1187-1203; Rexroad et al.,Molecular Reproduction and Development, 1997, 48, 238-245; Peng et al.,Biotechnology and Bioengineering, 1996, 50, 479-492; Grigoriev et al.,Analytical Biochemistry, 1996, 236, 250-254; Sanchez et al., J ImmunolMethods, 1991, 145, 193-197; Butcher et al., J Immunol Methods, 1988,107, 245-251; Long et al., J Immunol Methods, 1986, 86, 89-93; Shneyouret al., Plant Science Letters, 1984, 33, 293-302; Pintus et al., JImmunol Methods, 1983, 61, 195-200; Brodin et al., J ImmunologicalMethods, 1983, 60(1-2),1-7). Here, adherent feeder cells are laid out ina culture vessel or on a carrier as a single layer of cells (monolayer)and the cells to be cultivated are grown on this monolayer. As thefeeder cells have lost their capacity for further growth (eithernaturally or by artificial means) the cells which are to be cultivatedcan replicate without being overgrown by the feeder cells. Both thefeeder cells and the cells to be cultivated are adherently growingcells.

By contrast according to another embodiment the present invention makesit possible to replicate cells growing in suspension, either usingadherent autologous feeder cells or, in another preferred embodiment,using autologous feeder cells which are also kept in suspension. The useof autologous feeder cells kept in suspension is particularly preferredif both the feeder cell(s) and the cell(s) to be cultivated originatefrom the same basic cell, for example if both cells were originallycells which had adapted to growth in suspension. Thus, the presentinvention also relates to a method of replicating/cloning the mammaliancells described above, characterised in that the cells to be cultivatedare deposited, cultivated and replicated in the presence of autologousfeeder cells kept in suspension. Particularly preferred is acorresponding process which is characterised in that the cell(s) to becultivated is or are cell(s) adapted to growth in suspension. Alsopreferred in this connection is a corresponding method ofreplicating/cloning mammalian cells, characterised in that thedepositing of the cells and the replication of the deposited mammaliancells are carried out in a serum-free and/or protein-free and/orchemically defined suspension culture.

The number of autologous feeder cells to be used in thereplication/cloning of the mammalian cells described here dependsfundamentally on the nature of the mammalian cell which is to bereplicated and cloned and can be determined by simple titrationexperiments for each type of cell. The methods of replicating/cloningmammalian cells according to the invention are carried out for examplein the presence of at least more than 100 autologous feeder cells per mlof medium, preferably in the presence of 100 to 200,000 autologousfeeder cells per ml of medium. In another preferred embodiment thereplication/cloning of the mammalian cells is carried out in thepresence of 500 to 50,000 autologous feeder cells per ml of medium. Evenmore preferred is a process wherein the replication/cloning of themammalian cells is carried out in the presence of 500 to 10,000autologous feeder cells per ml medium, preferably in the presence of2,000 to 10,000 autologous feeder cells per ml medium.

In accordance with the definition of the term “autologous feeder cells”the present invention relates to methods of replicating/cloningmammalian cells, characterised in that hamster cells, preferably of thesubfamily Cricetinae, more preferably of the genus Cricetulus orMesocricetus, are used as feeder cells when the deposited mammaliancells which are to be replicated/cloned are CHO or BHK cells and mousemyeloma cells are used as feeder cells when the deposited mammaliancells which are to be replicated/cloned are NS0 cells. Also preferred isa process for replicating/cloning mammalian cells, characterised in thatCHO cells are used as feeder cells when the deposited mammalian cellswhich are to be replicated/cloned are CHO cells, in that BHK cells areused as feeder cells when the deposited mammalian cells which are to bereplicated/cloned are BHK cells, and in that NS0 cells are used asfeeder cells when the deposited mammalian cells which are to bereplicated/cloned are NS0 cells.

In the event that the deposited mammalian cells which are to becultivated are cultivated in the presence of a selection agent, it isappropriate to use autologous feeder cells which also have the selectionmarker gene which confers resistance, as this can prevent the feedercells from dying off too quickly in the presence of the selection agent.Accordingly, the present invention also relates to methods of cloningcells, particularly the above-mentioned hamster or mouse myeloma cells,characterised in that fewer than five, for example four, three, two, orone (1) of these mammalian cell(s) are deposited in a culture vessel inthe presence of autologous feeder cells under serum-free conditions andis or are cultivated and replicated under serum-free conditions, theautologous feeder cells and the deposited mammalian cell(s) each havingat least one selection marker gene which confers resistance to aselection agent, and at least the replication of the mammalian cell(s)to be cloned takes place under serum-free conditions in the presence ofsaid selection agent to which both the feeder cell and the mammaliancell to be cloned are resistant.

The autologous feeder cells may be produced for example by irradiatingwith a radioactive source of radiation, for example by irradiation withthe caesium isotope 137 (¹³⁷Cs). Irradiation with an energy dose ofbetween 1 and 1,000 Gy is advantageous for the methods ofreplicating/cloning mammalian cells in the presence of autologous feedercells as described here. It is particularly advantageous to use anenergy dose of between 10 and 500 Gy, more advantageously between 20 and200 Gy. In connection with the cloning of CHO cells it has been foundthat the use of autologous feeder cells, preferably CHO cells, isbeneficial and leads to a high level of cloning efficiency afterirradiation with an energy dose of between 1 and 500 Gy. It has provedparticularly advantageous to use autologous feeder cells which have beenirradiated with an energy dose of between 20 and 100 Gy, preferablyabout 50 Gy. Theoretically, the optimum energy dose for each cell can bedetermined experimentally by treating feeder cells with differentoverall doses of radiation and determining the cloning efficiency as afunction of the dose of radiation, analogously to the method describedin the examples. In addition to gamma radiation with ¹³⁷Cs and ⁶⁰Co(cobalt isotope 60) treatment with UV radiation, electron radiation,radioactive radiation, neutron radiation and microwave radiation arealso suitable, for example.

The feeder cells may be used directly or after cryopreservation, forexample in liquid nitrogen, in one of the methods of replicating/cloningmammalian cells according to the invention. Processes for cryopreservingmammalian cells are known in the art and are described by way of examplein Freshney (editor), Animal Cell culture—a practical approach,IRL-Press 1986, pages 73-78, the contents of which are herebyincorporated by reference.

The present processes are suitable for replicating the depositedmammalian cells up to a density of 1×10⁵ to 4×10⁶/ml medium in theculture vessel in which they were originally deposited. Preferably thefirst passaging takes place at a cell density of 2×10⁵ to 8×10⁵/ml ofmedium, particularly at a cell density of 2×10⁵ to 5×10⁵/ml of medium.

The methods of replicating/cloning the mammalian cells according to theinvention described here are characterised by a high level of recloningefficiency, which means that the present invention relates to processesfor replicating/recloning mammalian cells, characterised in that thecloning efficiency is at least 10%, preferably at least 20%, morepreferably at least 30% and even more preferably at least 40%. Accordingto a particularly preferred embodiment the present invention relates tomethods of replicating/recloning mammalian cells characterised in thatthe recloning efficiency is at least 50%, preferably at least 60% andparticularly preferably at least 70% and even more preferably at least80%.

According to the Examples described herein a recloning efficiency of(including) more than 65% was obtained for CHO cells. Thus, the presentinvention also relates to methods of replicating/recloning CHO cellswhich are characterised in that the recloning efficiency in therecloning of deposited CHO cells is from 10 to more than 65%, preferablymore than 20%, most preferably more than 30%, more preferably more than40%, even more preferably more than 50%, particularly more than 60%.However, the present invention also relates to processes with somewhatlower recloning efficiencies for the cell types specified.

Moreover, the present invention relates to methods of preparing one ormore products (polypeptides, proteins, nucleic acids, etc.), preferablyrecombinant products, in cells which are replicated/recloned accordingto one of the methods described above. The prerequisite is that the cellin question contains one or more genes of interest which code(s) for oneor more products to be prepared. Preferably the cell in question is aCHO, BHK or NS0 cell and derivatives/descendants of these cell lines.However, it may also be any other cell, e.g. one of the cells listed inTable 1.

The gene(s) of interest to be produced may be genes which occurnaturally in the host cell or they may be genes artificially introducedinto the cells. By definition each sequence or gene introduced into acell is referred to as a “heterologous sequence” or “heterologous gene”in relation to this cell, even if the sequence or gene to be introducedis identical to an endogenous sequence or an endogenous gene of thecell. For example, a hamster actin gene which is introduced into ahamster cell is by definition a heterologous gene. If this heterologousgene codes for a gene of interest it is also referred to as a“heterologous gene of interest”.

A heterologous gene of interest may be introduced into the cell byvarious methods, for example by viral transformation, transfection ormicroinjection. The heterologous gene of interest may be introduced intothe cell as linear DNA or as part of an expression vector. A number ofeukaryotic expression vectors are known which allow multiple cloningsites for the insertion of one or more heterologous genes and theirexpression. Commercial suppliers include among others companies such asStratagene, La Jolla, Calif., USA; Invitrogen, Carlsbad, Calif., USA;Promega, Madison, Wis., USA or BD Biosciences Clontech, Palo Alto,Calif., USA. The transfection of the cells with a DNA or an expressionvector which code(s) for one or more genes of interest is carried out byconventional methods as described for example in Sambrook et al., 1989or Ausubel et al., 1994. Suitable methods of transfection include forexample liposome-mediated transfection, calcium phosphateco-precipitation, electroporation, polycation—(e.g. DEAEdextran)-mediated transfection, protoplast fusion, microinjection andviral infections. Preferably, stable transfection is carried out inwhich the DNA molecules are either integrated into the genome of thehost cell or an artificial chromosome/minichromosome, or are episomallycontained in stable manner in the host cell. The transfection methodwhich gives the optimum transfection frequency and expression of one ormore heterologous genes of interest in the host cell in question ispreferred.

The heterologous gene of interest is usually functionally linked to apromoter which enables the transcription of the gene of interest, and toother regulatory elements which allow transcription and translation(expression) of the gene of interest or increase its efficiency.

The term “promoter” denotes a polynucleotide sequence which enables andcontrols transcription of the genes or sequences functionally linked toit. A promoter contains recognition sequences for binding RNA polymeraseand the initiation site for transcription (transcription initiationsite). In order to express a desired sequence in a certain cell type ora host cell a suitable functional promoter must be chosen. The skilledman will be familiar with a variety of promoters from various sources,including constitutive, inducible and repressible promoters. They aredeposited in databanks such as GenBank, for example, and may be obtainedas separate elements or elements cloned within polynucleotide sequencesfrom commercial or individual sources. In inducible promoters theactivity of the promoter may be reduced or increased in response to asignal. One example of an inducible promoter is the tetracycline (tet)promoter. This contains tetracycline operator sequences (tetO) which canbe induced by a tetracycline-regulated transactivator protein (tTA). Inthe presence of tetracycline the binding of tTA to tetO is inhibited.Examples of other inducible promoters are the jun, fos, metallothioneinand heat shock promoter (see also Sambrook, J., Fritsch, E. F. &Maniatis, T., Molecular Cloning: A Laboratory Manual Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989; Gossen, M. et al., Curr OpiBiotech 1994, 5, 516-520). Of the promoters which are particularlysuitable for high expression in eukaryotes, there are for example theubiquitin/S27a promoter of the hamster (WO 97/15664), SV 40 earlypromoter, adenovirus major late promoter, mouse metallothionein-Ipromoter, the long terminal repeat region of Rous Sarcoma Virus and theearly promoter of human Cytomegalovirus. Examples of other heterologousmammalian promoters are the actin, immunoglobulin or heat shockpromoter(s).

For example, the promoter may be functionally linked to enhancersequences in order to increase the transcriptional activity. For this,one or more enhancers and/or several copies of an enhancer sequence maybe used, e.g. a CMV or SV40 enhancer.

The term enhancer denotes a polynucleotide sequence which in the cislocation acts on the activity of a promoter and thus stimulates thetranscription of a gene functionally connected to this promoter. Unlikepromoters the effect of enhancers is independent of position andorientation and they can therefore be positioned in front of or behind atranscription unit, within an intron or even within the coding region.The enhancer may be located both in the immediate vicinity of thetranscription unit and at a considerable distance from the promoter. Itis also possible to have a physical and functional overlap with thepromoter. The skilled artisan will be aware of a number of enhancersfrom various sources (and deposited in databanks such as GenBank, e.g.SV40 enhancers, CMV enhancers, polyoma enhancers, adenovirus enhancers)which are available as independent elements or elements cloned withinpolynucleotide sequences (e.g. deposited at the ATCC or from commercialand individual sources). A number of promoter sequences also containenhancer sequences such as the frequently used CMV promoter. The humanCMV enhancer is one of the strongest enhancers identified hitherto. Oneexample of an inducible enhancer is the metallothionein enhancer, whichcan be stimulated by glucocorticoids or heavy metals.

Basically, the regulatory elements include promoters, enhancers,termination and polyadenylation signals and other expression controlelements. Both inducible and constitutively regulatory sequences areknown for the various cell types. “Transcription-regulatory elements”generally comprise a promoter upstream of the gene sequence to beexpressed, transcription initiation and termination sites and apolyadenylation signal.

The term “transcription initiation site” refers to a nucleic acid in theconstruct which corresponds to the first nucleic acid which isincorporated in the primary transcript, i.e. the mRNA precursor. Thetranscription initiation site may overlap with the promoter sequences.

The term “transcription termination site” refers to a nucleotidesequence which is normally at the 3′ end of the gene of interest or ofthe gene section which is to be transcribed, and which brings about thetermination of transcription by RNA polymerase.

The “polyadenylation signal” is a signal sequence which causes cleavageat a specific site at the 3′ end of the eukaryotic mRNA andposttranscriptional incorporation of a sequence of about 100-200 adeninenucleotides (polyA tail) at the cleaved 3′-end. The polyadenylationsignal comprises the sequence AATAAA about 10-30 nucleotides upstream ofthe cleavage site and a sequence located downstream. Variouspolyadenylation elements are known such as tk polyA, SV40 late and earlypolyA or BGH polyA (described for example in U.S. Pat. No. 5,122,458).

“Translation regulatory elements” comprise a translation initiation site(AUG), a stop codon and a polyA signal for each polypeptide to beexpressed. For optimum expression it may be advisable to remove, add orchange 5′- and/or 3′-untranslated regions of the nucleic acid sequencewhich is to be expressed, in order to eliminate any potentiallyunsuitable additional translation initiation codons or other sequenceswhich might affect expression at the transcription or expression level.In order to promote expression, ribosomal consensus binding sites mayalternatively be inserted immediately upstream of the start codon. Inorder to produce a secreted polypeptide the gene of interest usuallycontains a signal sequence which codes for a signal precursor peptidewhich transports the synthesised polypeptide to and through the ERmembrane. The signal sequence is often but not always located at theamino terminus of the secreted protein and is cleaved by signalpeptidases after the protein has been filtered through the ER membrane.The gene sequence will usually but not necessarily contain its ownsignal sequence. If the native signal sequence is not present aheterologous signal sequence may be introduced in known manner. Numeroussignal sequences of this kind are known to the skilled artisan anddeposited in sequence databanks such as GenBank and EMBL.

Gene products of interest may include proteins/polypeptides, e.g.antibodies, enzymes, cytokines, lymphokines, adhesion molecules,receptors and the derivatives or fragments thereof, but are notrestricted thereto. Generally, all polypeptides which act as agonists orantagonists and/or have therapeutic or diagnostic applications are ofvalue.

The term “polypeptides” is used for amino acid sequences or proteins andrefers to polymers of amino acids of any length. This term also includesproteins which have been modified post-translationally by reactions suchas glycosylation, phosphorylation, acetylation or protein processing.The structure of the polypeptide may be modified, for example, bysubstitutions, deletions or insertions of amino acids and fusion withother proteins while retaining its biological activity.

Examples of proteins are insulin, insulin-like growth factor (IGF-I orIGF-II); human growth hormone (hGH) and other growth factors such as forexample VEGF, EGF, TGF, for example TGF alpha and beta, including β1,β2, β3, β4 and β5; tissue plasminogen activator (tPA); erythropoietin(EPO); thrombopoietin (TBO); cytokines, for example interleukins (IL)such as IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12,IL13, IL14, IL15, IL16, IL17, IL18; interferon (IFN)-alpha, -beta,-gamma, -omega or -tau, tumour necrosis factor (TNF) such as TNF-alpha,-beta or -gamma, CD40-ligand, Apo2-ligand/TRAIL, DR4, DR5, DcR1, DcR2,DcR3, OPG, Fas ligand; GCSF; GMCSF; MCSF; MCP1 and VEGF. Other examplesare clotting factors such as factor VII, factor VIII, factor IX, vonWillebrands factor; anticoagulant factors such as protein C;enekephalinase; RANTES (regulated on activation normally T-cellexpressed and secreted); human macrophage inflammatory protein(MIP-1-alpha); (human) serum albumin, cell adhesion molecules, such asLFA1, Mac1, p150.95, VLA4, ICAM1, ICAM2, ICAM3, VCAM, or αV/β3 integrinincluding α or β subunits; blood group antigens; flk2/3 receptor; OBreceptor; mlp receptor; CTLA4; Apo2L receptors such as for example Apo2;Transforming Growth Factor (TGF); CD proteins, T-cell receptors; viralantigens such as for example gp120 of HIV; tumour associated antigenssuch as for example HER2, HER3 or HER4 receptor, rheumatoid factors, forexample NGF6 or PDGF; Relaxin-A or -B chain; gonadropin;gonadropin-associated peptide; inhibin; activin; a cytotoxicT-lymphocyte-associated antigen (CTLA) or neurotophin factors such asfor example BDNF, neurotrophin-3, -4, -5 or -6.

Other examples are monoclonal, polyclonal, multispecific and singlechain antibodies and fragments thereof such as for example Fab, Fab′,F(ab′)₂, Fc and Fc′ fragments, light (L) and heavy (H) immunoglobulinchains and the constant, variable or hypervariable regions thereof aswell as Fv and Fd fragments (Chamov, S. M. et al., Antibody FusionProteins, Wiley-Liss Inc., 1999). The antibodies may be of human ornon-human origin. Humanised and chimeric antibodies are also possible.

Fab fragments (fragment antigen binding=Fab) consist of the variableregions of both chains which are held together by the adjacent constantregions. They may be produced for example from conventional antibodiesby treating with a protease such as papain or by DNA cloning. Otherantibody fragments are F(ab′)₂ fragments which can be produced byproteolytic digestion with pepsin.

By gene cloning it is also possible to prepare shortened antibodyfragments which consist only of the variable regions of the heavy (VH)and light chain (VL). These are known as Fv fragments (fragmentvariable=fragment of the variable part). As covalent binding via thecystein groups of the constant chains is not possible in these Fvfragments, they are often stabilised by some other method. For thispurpose the variable region of the heavy and light chains are oftenjoined together by means of a short peptide fragment of about 10 to 30amino acids, preferably 15 amino acids. This produces a singlepolypeptide chain in which VH and VL are joined together by a peptidelinker. Such antibody fragments are also referred to as single chain Fvfragments (scFv). Examples of scFv antibodies are known and described,cf. for example Huston et al. (Huston, C. et al., Proc Natl Acad Sci USA1988, 85 (16), 5879-5883).

In past years various strategies have been developed for producingmultimeric scFv derivatives. The intention is to produce recombinantantibodies with improved pharmacokinetic properties and increasedbinding avidity. In order to achieve the multimerisation of the scFvfragments they are produced as fusion proteins with multimerisationdomains. The multimerisation domains may be, for example, the CH3 regionof an IgG or helix structures (“coiled coil structures”) such as theLeucine Zipper domains. In other strategies the interactions between theVH and VL regions of the scFv fragment are used for multimerisation(e.g. dia-, tri- and pentabodies).

The term diabody is used in the art to denote a bivalent homodimericscFv derivative. Shortening the peptide linker in the scFv molecule to 5to 10 amino acids results in the formation of homodimers bysuperimposing VH/VL chains. The diabodies may additionally be stabilisedby inserted disulphite bridges. Examples of diabodies can be found inthe literature, e.g. in Perisic et al. (Perisic, O. et al., Structure1994, 2, 1217-1226).

The term minibody is used in the art to denote a bivalent homodimericscFv derivative. It consists of a fusion protein which contains the CH3region of an immunoglobulin, preferably IgG, most preferably IgG1, asdimerisation region. This connects the scFv fragments by means of ahinge region, also of IgG, and a linker region. Examples of suchminibodies are described by Hu et al. (Hu, S. et al., Cancer Res. 1996,56 (13), 3055-3061).

The term triabody is used in the art to denote a trivalent homotrimericscFv derivative (Kortt, A. A. et al., Protein Engineering 1997, 10 (4),423-433). The direct fusion of VH-VL without the use of a linkersequence leads to the formation of trimers.

The fragments known in the art as mini antibodies which have a bi-, tri-or tetravalent structure are also derivatives of scFv fragments. Themultimerisation is achieved by means of di-, tri- or tetrameric coiledcoil structures (Pack, P. et al., Biotechnology 1993, 11, 1271-1277 andPack, P. et al., J Mol Biol 1995, 246 (11), 28-34; Lovejoy, B. et al.,Science 1993, 259, 1288-1293).

For selecting transfected cells these may additionally be transfectedwith one or more selectable marker genes. The literature describes alarge number of selectable marker genes including bifunctional(positive/negative) markers (see for example WO 92/08796 and WO94/28143). Examples of selectable markers which are usually used ineukaryotic cells include the genes for aminoglycoside phosphotransferase(APH), hygromycine phosphotransferase (HYG), dihydrofolate reductase(DHFR), thymidine kinase (TK), glutamine synthetase, asparaginsynthetase and genes which confer resistance to neomycin (G418),puromycin, histidinol D, bleomycin, phleomycin and zeocin. These genesmay be introduced into the cell together with the gene of interest orseparately. Preferably they are also introduced into the cells by meansof expression vectors. Correspondingly modified cells may be cultivatedin the presence of one or more suitable selecting agents whichselectively prefer cells in growth which contain and express acorresponding selectable marker gene.

It is also possible to select transfected cells byfluorescence-activated cell sorting (FACS), for example using bacterial6-galactosidase, cell surface markers or fluorescent proteins. Thefluorescent proteins also allow FACS-based isolation of individualmammalian cells. Correspondingly detected cells can automatically bedeposited in culture vessels as single cells or as a plurality of cells,e.g. using a laser, e.g. an argon laser (488 nm) and for example with aFlow Cytometer fitted with an Autoclone unit (Coulter EPICS Altra,Beckman-Coulter, Miami, Fla., USA). According to a preferred embodimentonly one (1) or at most two cells are deposited in a cell culture dishcontaining autologous feeder cells in this way. It is particularlyadvantageous to deposit only one (1) individual cell. In addition,sorting may be done using magnetic beads. For this the cells arelabelled, for example, using antibodies coupled to magnetic beads. Thisenables the cells to be sorted according to specific properties.

It is particularly advantageous to carry out FACS-based isolation ofcell clones which have been deposited according to one of the processesdescribed here and which co-express a fluorescent protein and a gene ofinterest. Preferably, the expression of the fluorescent protein and thegene of interest are functionally linked to each other. Such afunctional link consists, for example, of the two genes being arrangedclose together, so that the expression rates of the two genes arecorrelated, e.g. after transient or stable transfection of a host cell.Such functional linking may also be obtained, for example, by the use ofso-called IRES elements (internal ribosome entry site) or by RNAsplicing, the two genes (gene of interest and gene of the fluorescentprotein) being synthesised as bicistronic mRNA. In this way there is adirect correlation between the expression rate of the fluorescentprotein and the gene of interest. The corresponding cell clones whichexhibit high expression of fluorescent protein also have a highexpression rate of the gene of interest, as a result of the functionallinking.

The fluorescent protein may be, for example, a green, bluish-green,blue, yellow or other coloured fluorescent protein. One particularexample is green fluorescent protein (GFP) obtained from Aequoreavictoria or Renilla reniformis and mutants developed from them; see forexample Bennett, R. P. et al., BioTechniques 1998, 24, 478-482; Chalfie,M. et al., Science 1994, 263, 802-805; WO 01/04306 and the literaturecited therein. Other fluorescent proteins and genes coding for them aredescribed in WO 00/34318, WO 00/34326, WO 00/34526 and WO 01/27150 whichare incorporated herein by reference. These fluorescent proteins arefluorophores of non-bioluminescent organisms of the species Anthozoa,for example Anemonia majano, Clavularia sp., Zoanthus sp. I, Zoanthussp. II, Discosoma striata, Discosoma sp. “red”, Discosoma sp. “green”,Discosoma sp. “Magenta”, Anemonia sulcata. The fluorescent proteins usedmay consist of the wild-type proteins, natural or genetically engineeredmutants and variants, fragments, derivatives or variants thereof whichhave for example been fused with other proteins or peptides. Themutations used may for example alter the excitation or emissionspectrum, the formation of chromophores, the extinction coefficient orthe stability of the protein. Moreover, the expression in mammaliancells or other species can be improved by codon optimisation. Accordingto the invention the fluorescent protein may also be used in fusion witha selectable marker, preferably an amplifiable selectable marker such asdihydrofolate reductase (DHFR), for example.

The selection step can be carried out on cell populations or with cellpopulations/cell clones which have been pre-sorted. One or more,preferably one (1), two, three or four cells may be deposited per cellculture vessel. Preferably, the cells are deposited in serum-freemedium, most preferably in chemically defined medium, in the presence ofautologous feeder cells. Suitable media and methods according to theinvention for depositing cells using autologous feeder cells arediscussed in detail elsewhere in this application. Basically, two ormore sorting steps may be carried out, and between the separate sortingsteps the cells are cultivated and replicated over a particular lengthof time, e.g. about two weeks, as pools, in a suitable medium.

In order to produce one or more gene products of interest in therecloned cells the recloned cells are preferably grown in a serum-freeculture medium and in suspension culture under conditions which allowexpression of the gene of interest. If for example the gene of interestis under the control of a constitutive promoter, there is no need to addspecial inducers. If the expression of the gene of interest is under thecontrol of an inducible promoter, for example, a corresponding inducermust be added to the cell culture medium in a sufficient but non-toxicconcentration. The cells can be expanded as desired by multiplesubpassaging and transferred into suitable cell culture vessels. Thegene product(s) is or are produced as either a cellular, membrane-boundor secretory product.

The product of interest is preferably obtained from the cell culturemedium as a secreted gene product. If a protein or polypeptide isexpressed without a secretion signal, however, the gene product may alsobe isolated from cell lysates. In order to obtain a pure homogeneousproduct which is substantially free from other recombinant proteins andhost cell proteins, conventional purification procedures are carriedout. First of all, cells and cell debris are frequently removed from theculture medium or lysate. The desired gene product can then be freedfrom contaminating soluble proteins, polypeptides and nucleic acids,e.g. by fractionation on immunoaffinity and ion exchange columns,ethanol precipitation, reversed phase HPLC or chromatography onSephadex, silica or cation exchange resins such as DEAE. Methods whichresult in the purification of a heterologous protein expressed byrecombinant host cells are known to the skilled man and described in theliterature, e.g. by Harris et al. (Harris et al., Protein Purification:A Practical Approach, Pickwood and Hames, eds., IRL Press, Oxford, 1995)and Scopes (Scopes, R., Protein Purification, Springer Verlag, 1988).

In another aspect the present invention therefore relates to a method ofpreparing one or more products in mammalian cells under serum-freeconditions, characterised in that (i) mammalian cells contain a gene ofinterest which codes for a protein of interest; (ii) the mammalian cellsare grown under serum-free conditions which allow replication of themammalian cells; (iii) in each case fewer than five, preferably four,three, two or one (1) of these mammalian cell(s) are deposited in a cellculture vessel under serum-free conditions; (iv) the suitably depositedmammalian cells are replicated in the presence of autologous feedercells under serum-free conditions; (v) the replicated cells arecultivated under serum-free conditions in which the gene of interest isexpressed; and (vi) the gene product is then isolated from the cells orculture supernatant and purified. According to a preferred embodiment ofthis process, in point (iii) only one (1) or two (2) of the mammaliancells are deposited in each cell culture vessel. Particularly preferredis a process in which only one (1) single mammalian cell is deposited inpoint (iii). The cell deposition may be manual or automated, e.g. usingFACS-based cell sorting. According to another preferred embodiment themammalian cell is a transfected mammalian cell into which the gene ofinterest has been introduced. Accordingly, the present invention alsorelates to a method of preparing recombinant gene products,characterised in that before step (i) of the process described above themammalian cells are transfected with a nucleic acid which at least codesfor a gene of interest. Stable transfection of the correspondingmammalian cell is preferred.

As already mentioned, the present invention also relates to FACS-basedsorting of individual mammalian cells and the deposition of single ormultiple mammalian cells, preferably fewer than 5, more preferably 4, 3,2 or 1 mammalian cell(s) which express a protein of interest, the cellsorting and cell deposition preferably taking place as a function of theexpression rate of the fluorescent protein co-expressed in the mammaliancell, the expression of which is functionally linked with the expressionof the protein of interest. Accordingly, the present invention alsorelates to a method of producing a recombinant protein in mammaliancells under serum-free conditions, characterised in that i) themammalian cells are transfected with a gene which codes for a protein ofinterest; ii) the mammalian cells are transfected with a gene whichcodes for a fluorescent protein, the expression of the gene which codesfor a fluorescent protein preferably being functionally linked to theexpression of the gene of interest; iii) the transfected mammalian cellsare grown under serum-free conditions which allow replication of thetransfected cells and expression of at least the fluorescent protein;iv) in each case fewer than 5, preferably 4, 3, 2 or 1 (one) transfectedmammalian cell(s) is or are deposited in a cell culture vessel withautologous feeder cells under serum-free conditions after FACS-basedsorting, the FACS-based cell sorting being carried out on the basis ofthe expression rate of the fluorescent protein; v) the correspondinglydeposited cells are replicated in the presence of the autologous feedercells under serum-free conditions; vi) the replicated cells are grownunder serum-free conditions in which at least the gene of interest isexpressed; and vii) the gene product (of the gene of interest) is thenisolated from the cells or from the culture supernatant and purified.According to a particularly preferred embodiment, in step iv) only one(1) individual cell is deposited and replicated per culture vessel.

Preferably, only the cells which belong to the 20% of cells with thehighest expression rate of fluorescent protein are sorted out underpoint iv). In practice, this means that the brightest 20% of thefluorescent cells are sorted out (20% most fluorescent cells). Accordingto another preferred embodiment, only the brightest 5%, preferably thebrightest 3%, or only the brightest 1% of the fluorescent cells of acell mixture are sorted out. As shown in FIGS. 3 a and 3 b, this leadsto an enrichment of cell clones with a comparatively high expressionrate of the gene of interest. The FACS-based deposition of single cellsthus enables identification and replication of homogeneous cell cloneswhich have a comparatively high expression rate of a gene of interest,which in turn form the starting point for further optimising steps (e.g.gene amplification).

Also preferred is a corresponding method of producing one or morerecombinant products in recloned mammalian cells, characterised in thatthe mammalian cells are hamster or mouse myeloma cells, preferably CHO,BHK or NS0 cells, and derivatives/descendants of these cell lines.Another embodiment of this process is characterised in that the feedercells are adapted to serum-free medium and are non-adherently cultivatedcells. Also preferred in connection with the production processdescribed above is the use of hamster cells as feeder cells, if thedeposited mammalian cell(s) which are to be replicated/cloned are CHO orBHK cells, and the use of mouse myeloma cells as feeder cells, if thedeposited mammalian cell(s) which are to be replicated/cloned are NS0cells. Particularly preferred is a process which is characterised inthat CHO cells are used as feeder cells if the deposited mammaliancell(s) which are to be replicated/cloned are CHO cells, BHK cells areused as feeder cells if the deposited mammalian cell(s) which are to bereplicated/cloned are BHK cells and NS0 cells are used as feeder cellsif the deposited mammalian cell(s) which are to be replicated/cloned areNS0 cells.

The present invention for the first time provides hamster cells andmouse myeloma cells, preferably NS0 cells, as feeder cells. For thisreason the present invention also relates to the use of a hamster cellor a mouse myeloma cell of type NS0 as a feeder cell. Methods ofpreparing corresponding feeder cells are described in more detail in theExamples. The corresponding hamster or mouse myeloma feeder cells cantheoretically be prepared using chemical or physical methods known tothe skilled man, for example by treating with mitomycin C(azirino[2′,3′:3,4]pyrrolo[1,2-a]indole-4,7-dione,6-amino-8-[[(aminocarbonyl)oxy]methyl]-1,1a,2,8,8a,8b-hexahydro-8a-methoxy-5-methyl,[1aR(1a.alpha., 8.beta., 8a.alpha., 8b.alpha.)]-(9Cl) (Butcher et al., JImmunol Methods, 1988, 107, 245-251) or by irradiation with ¹³⁷Cs.According to a preferred embodiment the present invention thereforerelates to the use of chemically or physically inactivated feeder cells.The term “inactivated” means in this context that the cells arerestricted in their ability to divide and have preferably lost thisability but still remain vital. This means that the cells still retainmetabolic activities such as for example the synthesis and secretion ofgrowth factors for a certain period, preferably for at least one to twoweeks after inactivation. According to a preferred embodiment thecorresponding feeder cells are feeder cells which are adapted toserum-free culture conditions. According to a yet more preferredembodiment of the invention the feeder cells are CHO, BHK or NS0 cellsand the descendants/derivatives of these cell lines. Moreover, all thecells mentioned in this application may be used as feeder cells afterbeing suitably inactivated.

Furthermore, the present invention relates to compositions consisting ofa serum-free cell culture medium, fewer than five mammalian cellscapable of dividing, preferably four, three, two or one (1) mammaliancell(s) capable of dividing and feeder cells which are autologous withthe mammalian cells capable of dividing. According to a preferredembodiment of the present invention the corresponding compositioncontains only one (1) or two mammalian cells capable of dividing.According to another preferred embodiment the corresponding compositioncontains only one (1) mammalian cell capable of dividing.

Other preferred compositions contain as feeder cells hamster cells,preferably of the subfamily Cricetinae, most preferably of the genusCricetulus or Mesocricetus, if the mammalian cell(s) capable of dividingis or are CHO or BHK cells or derivatives/descendants thereof. Moreover,another preferred composition contains, as feeder cells, mouse cells,preferably of the subfamily Murinae, most preferably of the genus Mus,if the mammalian cell(s) capable of dividing is or are mouse hybridomacells, preferably NS0 cells or derivatives/descendants thereof.Particularly preferred compositions are characterised in that thecomposition contains CHO cells as feeder cells if the mammalian cell(s)capable of dividing is or are CHO cells, the composition contains BHKcells as feeder cells if the mammalian cell(s) capable of dividing is orare BHK cells, and the composition contains NS0 cells as feeder cells ifthe mammalian cell(s) capable of dividing is or are NS0 cells.

Theoretically, the invention also relates to compositions which enablefewer than 5, preferably 4, 3, 2, or 1 production-relevant hamstercell(s), such as for example CHO or BHK cell(s) and production-relevantmouse myeloma cells, such as for example NS0 cell(s) to be deposited andreplicated in the presence of feeder cells from mammals under serum-freeconditions.

Examples of serum-free, protein-free or chemically defined media includefor example the commercially obtainable media Ham's F12 (Sigma,Deisenhofen, DE), RPMI 1640 (Sigma), Dulbecco's Modified Eagle's medium(DMEM; Sigma), Minimal Essential medium (MEM; Sigma), Iscove's ModifiedDulbecco's medium (IMDM; Sigma), CDCHO (Invitrogen, Carlsbad, Calif.,USA), CHO-S-SFMII (Invitrogen), serum-free CHO medium (Sigma) andprotein-free CHO medium (Sigma). Each of these media can if desired besupplemented with various compounds such as hormones and/or other growthfactors (e.g. insulin, transferrin, epidermal growth factor,insulin-like growth factor), salts (e.g. sodium chloride, calcium,magnesium, phosphate), buffers (e.g. HEPES), nucleosides (e.g.adenosine, thymidine), glutamine, glucose or other equivalent nutrients,antibiotics and/or trace elements. If the replicable cells arerecombinant cells which express one or more selectable markers, one ormore suitable selection agents such as antibiotics may also be added tothe medium.

EXAMPLES Abbreviations ATCC American Type Culture Collection BHK BabyHamster Kidney

⁶⁰Co Cobalt isotope 60¹³⁷Cs Caesium isotope 137

CHO Chinese Hamster Ovary CMV Cytomegalovirus DE Germany DEAEDiethylaminoethyl DMSO Dimethylsulphoxide

DNA Desoxyribonucleic acidFACS Fluorescence-activated cell sorterFITC Fluorescein isothiocyanate

Gy Gray

HBSS Hank's balanced salt solutionHPLC High performance liquid chromatographymRNA Messenger ribonucleic acidNS0 Mouse hybridoma cellpolyA Polyadenylation sequenceSp2/0 Mouse hybridoma cell

SV40 Simian Virus No. 40 Methods 1. Cell Culturing

The cells CHO-DG44/dhfr^(−/−) (Urlaub, G. et al., Cell 1983, 33,405-412) were permanently cultivated as suspension cells in serum-freeCHO-S-SFMII medium (Invitrogen GmbH, Karlsruhe, DE) supplemented withhypoxanthine and thymidine in cell culture flasks at 37° C. in a humidatmosphere and 5% CO₂. The cell counts and viability were determinedwith a CEDEX Cell Counter (Innovatis, DE) or by trypan blue staining andthe cells were then seeded in a concentration of 1-3×10⁵/mL and passagedevery 2-3 days. For the single cell cloning recombinantCH₀-DG44/dhfr^(−/−) were used which express a fluorescent protein (forexample ZS-Green from Zoanthus sp.) or a fluorescent protein and a humanor humanised monoclonal antibody. Cloned recombinant cells were culturedanalogously to these cells. The medium used was again CHO-S-SFMII medium(Invitrogen GmbH, Karlsruhe, DE) without hypoxanthine and thymidine.

The BHK cells can be permanently cultivated as suspension cells inserum-free Opti Pro SFM medium (Invitrogen GmbH, Karlsruhe, DE) in cellculture flasks at 37° C. in a humid atmosphere under 5% CO₂. The cellcounts and viability can be determined with a CEDEX Cell Counter(Innovatis, DE) or by trypan blue staining and the cells are then seededin a concentration of 1-3×10⁵/mL and passaged every 2-3 days. Clonedcells are cultivated analogously to the BHK cells.

The NS0 cells can be permanently cultivated as suspension cells inserum-free hybridoma medium, animal component free medium (Sigma,Aldrich, St. Louis, USA) in cell culture flasks at 37° C. in a humidatmosphere at 5% CO₂. The cell counts and viability can be determinedwith a CEDEX Cell Counter (Innovatis, DE) or by trypan blue staining andthe cells are then seeded in a concentration of 1-3×10⁵/mL and run every2-3 days. Cloned cells are cultivated analogously to the NS0 cells.Hybridoma medium, animal component free medium (Sigma, Aldrich, St.Louis, USA) is used as the medium.

2. Preparation of Feeder Cells by Irradiation

Suspended CHO basic cells (non-transfected cell) growing serum- andprotein-free were centrifuged for 10 minutes at 180 g and adjusted to acell concentration of 1×10⁶/ml in HBSS (Hank's balanced salt solution).Then, the cells were irradiated with a radioactive radiation source(Cs137 radiator, Gammacell 2000, Messrs Molsgaard Medical A/S, Denmark)with an energy dose output of 4Gy/min. With an irradiation time ofbetween 5 min and 125 min, an energy dose of between 20 and 500 Gy wasobtained. After irradiation the cells were seeded into 96-wellmicrotitre plates at the rate of about 2000 cells/well (=culture vessel)in the CHO-S-SFMII medium specific for the cell and stored at about 37°C. and 5% CO₂ in an incubating chamber atmosphere. The process iscarried out in the same way with BHK and NS0 cells, the feeder cellsbeing kept or seeded in the medium specific for the cells.

3. Cryopreservation of Feeder Cells

The feeder cells thus produced can be cryopreserved at below −150° C.The cryopreservation is carried out in the cell culture medium inquestion using a programmable freezer (Consarctic BV25, Consarctic,Schöllkrippen, DE). 10% (v/v) DMSO is added to the media as acryoprotectant. The freezing rate between 0° C. and −20° C. is 1°C./min, then the temperature is lowered further at 0.4° C./min. Oncefreezing is complete the feeder cells are cryopreserved in liquidnitrogen in the gaseous phase.

4. Automated Cell Deposition

The automatic deposition of the cells (singly or in multiples) iscarried out with a Flow Cytometer fitted with an Argon Laser (488 nm)(Coulter EPICS Altra (Messrs Beckman-Coulter, Miami, Fla., USA) using anautoclone unit. The cells are centrifuged during the exponential growthphase and taken up in HBSS to a cell concentration of 1-1.5×10⁷/ml. Thenthe cells are sorted at a rate of 8000-12000 cells/second according totheir position in the scattered light using the “Hypersort Option”.Cells which express a fluorescent protein can alternatively be sortedaccording to their fluorescence intensity in relation to theintracellularly expressed fluorescent protein. The cells are depositedsingly in 96-well microtitre plates containing feeder cells. BHK cellsfor example are deposited in OptiPro SFM medium (Invitrogen GmbH,Karlsruhe, DE). The sorted NS0 cells are deposited for example inhybridoma medium, animal component free medium (Sigma, Aldrich, St.Louis, USA). In the sorting of CHO cells the cells were deposited inCHO-S-SFM-II (Invitrogen GmbH, Karlsruhe, DE). A recombinant CHO-DG-44cell was deposited which coexpressed a human or humanised monoclonalantibody as well as the ZS green from Zoanthus sp. The cell sorting wascarried out as described above with an argon laser at 488 nm.

5. Determining the Productivity of Recombinantly Expressed Gene Products(for the mAb-Expressing CHO-DGH-44 Used)

The antibody titre in the supernatants from stably transfected CHO-DG-44cells which express a human or humanised monoclonal antibody wasquantified by ELISA according to standard methods (Ausubel, F. M. etal., Current Protocols in molecular biology. New York: Greene PublishingAssociates and Wiley-Interscience. 1994 (updated)), using on the onehand a goat anti-human IgG Fc fragment (Dianova, Hamburg, DE) and on theother hand an AP-conjugated goat anti-human kappa light chain antibody(Sigma). The purified antibody was used as the standard. Theproductivities (pg/cell/day) were calculated according to the formulapg/((CtCo) t/In (CtCo)), where Co and Ct give the cell count on seedingor harvesting and t indicates the cultivation time.

Example 1 Influence of the Energy Dose During the Production of theFeeder Cells on the Recloning Efficiency of CHO-DG-44 Cells

In order to investigate the influence of the dose of energy during theproduction of the feeder cells on the recloning efficiency, theCHO-DG-44 cells were grown as described under Methods “Culturing of thecells”. The feeder cells were prepared as described in Methods“Preparation of feeder cells by irradiation” with an energy dose of 20Gy, 50 Gy, 100 Gy, 200 Gy and 500 Gy. After the irradiation the feedercells were seeded into 96-well microtitre plates with a cell count ofabout 2000 cells/well and stored in an incubating chamber atmosphere.Then, automated depositing of a single cell was carried out with arecombinant CHO-DG-44 cell which expressed a fluorescent protein, asdescribed under Methods “Automated single cell deposition”. One (1)individual cell was deposited on the feeder cells in each well. Thetarget value for the recloning efficiency was the number of positivewells, i.e. the well in which there were clones which had grown to forma cell population after an incubation period of three weeks. Therecloning efficiencies achieved were between 40 and 70% for therecloning of recombinant CHO-DG-44 cells (FIG. 1).

Example 2 Homogeneity of the Recloning of Antibody-Expressing CHO-DG-44Cells

In order to compare the homogeneity of the cell clones, recombinantantibody-expressing CHO-DG-44 cells were deposited and cloned on the onehand by the standard “limited dilution” method and on the other handdeposited and cloned by the single cell deposition method describedhere, in the presence of autologous feeder cells (FIG. 2). For this, sixcell pools of transfected antibody-expressing CHO-DG-44 cells werecultivated by the limited dilution method and parallel after theautomated single cell deposition, as described under “Culturing of thecells”, and then recloned as described under “Automated single celldeposition”. The clones thus produced were cultivated as described under“Culturing of the cells” and the product titre was determined over thecourse of three passages using the method “Determining the productivityof recombinantly expressed gene products”. The average obtained fromthese three passages was used to plot the graph.

Example 3 High Throughput Method for Generating High-Titre Cell Clonesby Combining the Expression of Fluorescent Proteins with FACS-Based CellSorting and FACS-Based Deposition of Single Cells

By transfecting CHO DG44 cells with an expression vector which codes thebicistronic expression of a product gene (recombinant antibody) and afluorescent protein (ZS Green from Zoanthus sp.), cell pools wereobtained which co-express both the antibody and the fluorescent protein.These cell pools were individually deposited and cultured in microtitreplates in the presence of autologous CHO DG44 feeder cells using themethod described above. The depositing of the clones was carried outusing three different criteria.

-   -   A.) Depositing all the living cells    -   B.) Depositing the 20% most strongly fluorescent cells    -   C.) Depositing the 5% most strongly fluorescent cells

The clones obtained were then transferred into a 24-well macrotitreplate and cultivated for three passages as described in “Cultivation ofthe cells”. At the end of each passage the titre of the antibody in thesupernatant was determined by the method described in “Determining theproductivity of recombinantly expressed gene products”. The averagevalue obtained from these three passages was used to plot the graph. Todo this, the number of clones obtained over defined titre categories wasplotted and matched to a normal distribution.

1-18. (canceled)
 19. A method of producing a protein in mammalian cellswhich contain a gene which codes for the protein under serum-freeconditions, comprising: (a) growing the mammalian cells which contain agene which codes for the protein under serum-free conditions which allowreplication of the mammalian cells; (b) depositing fewer than five ofthe mammalian cells in a cell culture vessel under serum-freeconditions; (c) replicating the suitably deposited mammalian cells inthe presence of autologous feeder cells under serum-free conditions; (d)cultivating the replicated cells under serum-free conditions in whichthe gene of interest is expressed; and (e) isolating and purifying thegene product from the cells or the culture supernatant.
 20. A method ofpreparing a recombinant protein in mammalian cells under serum-freeconditions, comprising: (a) transfecting the mammalian cells with a genewhich codes for a protein of interest; (b) growing the transfectedmammalian cells under serum-free conditions which allow replication ofthe transfected cells; (c) depositing fewer than 5 transfected mammaliancells in a cell culture vessel with autologous feeder cells underserum-free conditions; (d) replicating the deposited cells in thepresence of the autologous feeder cells under serum-free conditions; (e)growing the replicated cells under serum-free conditions in which thegene of interest is expressed; and (f) isolating and purifying the geneproduct from the cells or from the culture supernatant.
 21. The methodaccording to claim 19, wherein under step (b) only one (1) singlemammalian cell is deposited in each cell culture vessel.
 22. The methodaccording to claim 20, wherein under step (c) only one (1) singlemammalian cell is deposited in each cell culture vessel.
 23. The methodaccording to claim 19, wherein the mammalian cell deposited additionallycodes for a fluorescent protein and the mammalian cells deposited aredeposited by FACS-based cell sorting in a cell culture vessel underserum-free conditions.
 24. The method according to claim 20, wherein themammalian cell deposited additionally codes for a fluorescent proteinand the mammalian cells deposited are deposited by FACS-based cellsorting in a cell culture vessel under serum-free conditions.
 25. Themethod according to claim 19, wherein the mammalian cells are hamster ormouse myeloma cells.
 26. The method according to claim 20, wherein themammalian cells are hamster or mouse myeloma cells.
 27. The methodaccording to claim 25, wherein the hamster cells are CHO or BHK cells.28. The method according to claim 26, wherein the hamster cells are CHOor BHK cells.
 29. The method according to claim 25, wherein the mousemyeloma cells are NS0 cells.
 30. The method according to claim 26,wherein the mouse myeloma cells are NS0 cells.
 31. The method accordingto claim 19, wherein the feeder cells are non-adherently cultivatedcells adapted to serum-free medium.
 32. The method according to claim20, wherein the feeder cells are non-adherently cultivated cells adaptedto serum-free medium.
 33. The method according to claim 19, wherein thefeeder cells are hamster cells when the mammalian cells are CHO or BHKcells.
 34. The method according to claim 19, wherein the feeder cellsare mouse myeloma cells when the mammalian cell are NS0 cells.
 35. Themethod according to claim 20, wherein the feeder cells are hamster cellswhen the mammalian cells are CHO or BHK cells.
 36. The method accordingto claim 20, wherein the feeder cells are mouse myeloma cells when themammalian cell are NS0 cells.
 37. The method according to claim 33,wherein CHO cells are used as the feeder cells when the mammalian cellsare CHO cells and BHK cells are used as the feeder cells when themammalian cells are BHK cells.
 38. The method according to claim 33,wherein NS0 cells are used as the feeder cells when the mammalian cellsare NS0 cells.
 39. The method according to claim 34, wherein CHO cellsare used as the feeder cells when the mammalian cells are CHO cells andBHK cells are used as the feeder cells when the mammalian cells are BHKcells.
 40. The method according to claim 34, wherein NS0 cells are usedas the feeder cells when the mammalian cells are NS0 cells.
 41. Themethod according to claim 19, wherein the mammalian cells are replicatedin the presence of 100 to 200,000 feeder cells per ml of medium.
 42. Themethod according to claim 20, wherein the mammalian cells are replicatedin the presence of 100 to 200,000 feeder cells per ml of medium.
 43. Themethod according to claim 19, wherein the mammalian cells are cultivatedup to a cell density of 4×10⁶ cells/ml of medium.
 44. The methodaccording to claim 20, wherein the mammalian cells are cultivated up toa cell density of 4×10⁶ cells/ml of medium.
 45. The method according toclaim 25, wherein hamster cell is physically or chemically inactivated.46. The method according to claim 26, wherein hamster cell is physicallyor chemically inactivated.
 47. The method according to claim 25, whereinthe hamster cell is adapted to serum-free culture conditions.
 48. Themethod according to claim 26, wherein the hamster cell is adapted toserum-free culture conditions.
 49. The method according to claim 45,wherein the hamster cell is a CHO or BHK cell or a descendant or aderivative of the CHO or BHK cell.
 50. The method according to claim 46,wherein the hamster cell is a CHO or BHK cell or a descendant or aderivative of the CHO or BHK cell.
 51. The method according to claim 29,wherein the NS0 cell is physically or chemically inactivated.
 52. Themethod according to claim 30, wherein the NS0 cell is physically orchemically inactivated.
 53. The method according to claim 51, whereinthe NS0 cell is adapted to serum-free culture conditions.
 54. The methodaccording to claim 52, wherein the NS0 cell is adapted to serum-freeculture conditions.
 55. A composition consisting of a serum-free cellculture medium, fewer than five mammalian cells capable of dividing, andfeeder cells which are autologous to the mammalian cells capable ofdividing.
 56. The composition according to claim 55, wherein thecomposition contains only one or two mammalian cell(s) capable ofdividing in the culture medium.
 57. The composition according to claim55, wherein the composition contains hamster cells as feeder cells whenthe mammalian cells capable of dividing are CHO or BHK cells.
 58. Thecomposition according to claim 55, wherein the composition containsmouse myeloma cells as feeder cells when the mammalian cells capable ofdividing are NS0 cells.
 59. The composition according to claim 55,wherein the composition contains CHO cells as feeder cells when themammalian cells capable of dividing are CHO cells.
 60. The compositionaccording to claim 55, wherein the composition contains BHK cells asfeeder cells when the mammalian cells capable of dividing are BHK cells.61. The composition according to claim 55, wherein the compositioncontains NS0 cells as feeder cells when the mammalian cells capable ofdividing are NS0 cells.